In order to cost effectively produce biofuels from renewable plant biomass, all sugars, including all pentose and hexose sugars present in the raw lignocellulosic starting material, must be converted efficiently into the final products (1). The yeast, Saccharomyces cerevisiae, is an excellent host microbe for a range of industrial applications, from chemical and commodity production, to biofuel synthesis (2-4). However, S. cerevisiae does not readily uptake and use pentose sugars. This includes xylose, the most abundant pentose, and the second most abundant sugar next to glucose, found in biomass (5). While native xylose-utilizing organisms exist, they largely lack well-developed genetic tools for host engineering or exhibit low product and inhibitor tolerances. Therefore, it is important to engineer S. cerevisiae for more efficient xylose utilization, so that maximal carbon can be converted into biofuel.
Generating a yeast strain that utilizes xylose, especially in a glucose/xylose mix has been an object of extensive research for several decades (6). Great success has been achieved in boosting the native yeast utilization capability. Two approaches are now used routinely to provide for xylose utilization: overexpression of a heterologous xylose isomerase (XI) (7-11), and overexpression of the native or heterologous xylose reductase (XR) and xylitol dehydrogenase (XDH) (12, 13). Both pathways result in the transformation of xylose to xylulose, and benefit from additional overexpression of xylulokinase (XKS) to shunt the carbon into pentose-phosphate pathway (PPP) (14, 15). Further overexpression of genes encoding enzymes in the pentose-phosphate pathway, such as the transaldolase (TAL1) and the transketolase (TKL1), leads to further improvements in xylose assimilation rates (7, 16-18). Recently, it has also been shown that xylose utilization can be achieved via replacement of the native S. cerevisiae xylose utilization and PPP genes with those from the xylose-utilizing yeast Scheffersomyces stipites (19).
The improvements in intracellular xylose consumption have led to a bottleneck in xylose uptake (20). To date there has been no discovery of a sugar transporter that, in S. cerevisiae, allows for xylose uptake comparable to glucose uptake. S. cerevisiae has numerous monosaccharide transporters (HXT1-17 and GAL2), but all of them have greater specificity for hexose sugars. While a few of these (HXT1, 2, 4, 5, 7 and GAL2) can import xylose, they display rates of uptake so low that they cannot provide for growth on xylose (6, 21-25). Further, xylose uptake in these native transporters is repressed in the presence of glucose, limiting the use of these transporters in mixed sugar sources (26, 27).
Several strategies have been employed to tackle the issues with xylose transport. Much work has been devoted to bioprospecting and characterizing heterologous xylose-transporters in S. cerevisiae, resulting in the identification of several membrane proteins that can transport xylose (22, 28-33). These studies have shown that increasing xylose transport does increase utilization and final product formation, proving that xylose import is the limiting factor in utilization. However, these transporters have had limited efficacy either due to reduced growth rates, problems with substrate affinities, transport rates, or substrate inhibition.
Recently, a few studies have attempted to improve transport by engineering native transporters with encouraging results. Using a combination of bioinformatics, and mutagenesis, Young and colleagues, identified a xylose transport sequence motif, and were able to produce a mutant HXT7 strain that grew on xylose, but not glucose (34). Although this strain still showed glucose inhibition, another group was able to bypass this problem using growth to screen for glucose insensitivity (35). This approach resulted in the discovery of Gal2 and Hxt7 variants that bypass glucose inhibition. Unfortunately, the modifications that eliminated glucose repression also resulted in diminished uptake rates (Vmax). Though impressive, the resulting growth on xylose remained modest in both these studies and would benefit from further optimization.